SYNTHESIS OF DIMMER BLOCKS AND THEIR USE IN ASSEMBLING OLIGONUCLEOTIDES

Abstract: The invention provides methods for synthesizing dimer blocks (dinucleoside derivatives having modified internucleoside linkages) using simplified chemistries, novel dimer blocks, and methods of using dimer blocks to assemble oligonucleotides having modified internucleotide linkages.

Description: SYNTHESIS OF DIMMER BLOCKS AND THEIR USE IN ASSEMBLING OLIGONUCLEOTIDES BACKGROUND OF THE INVENTION Field Of The Invention The invention relates to the chemical synthesis of oligonucleotides. More particularly, the invention relates to the synthesis of oligonucleotides having modified internucleotide linkages. Summary Of The Related Art Since the discovery by Zamecnik and Stephenson, Proc. Natl. Acad. Sci. 75: 280-284 (1978), that synthetic oligonucleotides can inhibit Rous sarcoma virus replication, there has been great interest in the use of oligonucleotides and oligonucleotide analogs having modified internucleotide linkages to control gene regulation and to treat pathological conditions. Various methods have been developed for the synthesis of oligonucleotides for such purposes. Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72.: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 3_4: 3143-3179 (1978) discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 2j2: 1859-1862 (1981) discloses the use of deoxynucleoside phosphoramidites in polynucleot.ide synthesis. Agrawal and Zamecnik, U.S. Patent No. 5,149,798 (1992) discloses optimized synthesis of oligonucleotides by the H-phosphonate approach. Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28 . : 3539-3542 (1987) teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 2J3.: 3443 (1984) discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochemistry 27: 7237 (1988) discloses synthesis of oligonucleotide phosphoramidates using phoshoramidite chemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7083 (1988) discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry. Both phosphoramidite and H-phosphonate chemical syntheses are carried out on a solid support that is stored in a reaction vessel. The required reaction steps for coupling each nucleotide are detritylation, coupling, capping, and oxidation. For small scale (up to 1 μm ole) synthesis, the total time for the addition of one nucleotide is about 6 minutes. An oligonucleotide, 30-mer in length, can be assembled in 180 minutes. Under these conditions, synthesized oligonucleotides are chemically pure and biologically active. However, when oligonucleotides are synthesized on a larger scale (up to 1 mmole) , the time for addition of each nucleotide onto CPG is in the range of 50 to 60 minutes, requiring approximately 25 hours for assembling a 25-mer oligonucleotide. The increase in time is because of the volume of the solid support being used in synthesis. This increase in cycle time exposes the already assembled oligonucleotide sequence to all reaction steps (including dichloroacetic acid step, coupling step, oxidation step and capping step) for a longer time. This increase in total assembly time affects the yield as well as chemical and biological properties of the compound. The chemical and biological properties are mainly affected by depurination, base modifications, and the like. To reduce the effects of these problems, it is possible to synthesize oligonucleotides using dimeric or multimeric synthons, thereby reducing the number of cycles, and thus the time required for synthesizing oligonucleotides. To this end, several investigators have worked toward developing acceptable dimeric or multimeric synthon approaches. Khorana, Science 203: 614-625 (1979) introduced the concept of multimeric synthons, using a phosphodiester approach. Crea and Itakura, Proc. Natl. Acad. Sci. USA 75: 5765-5769 (1978); Reese, Tetrahedron Lett. 3_4: 3143-3179 (1978); and Ohtsuka et al. , Nucleic Acids Res. 1J): 6553-6570 (1982) all disclose use of dimeric or multimeric synthons in a phosphotriester approach. Kumar and Poonian, J. Org. Che . .49 . : 4905-4912 (1984) ; and Wolter et al., Nucleosides and Nucleotides 5_: 65-77 (1986) disclose phosphoramidite synthesis of oligonucleotide phosphodiesters using dimeric synthons. Marugg et al., Nucleic Acids Res. .12.: 9095-9110 (1984) teaches use of a dinucleotide thiophosphotriester to produce oligonucleotides containing one phosphorothioate linkage. Connolly et al.. Biochemistry 22.' 3443 (1984) ; and Cosstick and Eckstein, Biochemistry 24 . : 3630 (1985) disclose addition of one dinucleotide phosphorothioate to a growing oligonucleotide chain using a phosphoramidite approach. Brill and Caruthers, Tetrahedron Lett. 2j8: 3205 (1987) discloses synthesis of thymidine dinucleotide methylphosphonothioates. Roelen et al.. Nucleic Acids Res. .16: 7633-7645 (1988) discloses use of the dimeric nucleotide methylphosphonothioate TA to introduce a single methylphosphonothioate linkage into a short hexameric oligonucleotide in a solution phase reaction. The foregoing studies represent progress in the development of methods for synthesizing oligonucleotides using dimeric or multimeric synthons. Unfortunately, certain deficiencies remain extant. For example, the existing chemistries for producing dimeric nucleotides containing phosphorothioate or methyl- phosphonothioate internucleotide linkages are highly complex. Thus, only two methylphosphonothioate-containing dimers (TT and TA) have been reported to date. In addition, none of the existing dimeric synthons have been successfully used to synthesize oligonucleotides having exclusively alkylphosphonate, phosphoramidate, phosphorothioate, or alkylphosphonothioate internucleotide linkages. There is, therefore, a need for new methods employing simplified chemistries for the synthesis of dimeric synthons, as well as for new methods for using such dimeric synthons to assemble oligonucleotides. BRIEF SUMMARY OF THE INVENTION The invention provides new methods for producing dimeric nucleotide synthons, hereafter called "dimer blocks", having modified internucleotide linkages, and preferably alkylphosphonate phosphoramidate, phosphorothioate or alkylphosphonothioate internucleotide linkages. According to this aspect of the invention, synthesis of dimer blocks proceeds in a single pot solution phase reaction, regardless of the type of internucleotide linkage in the dimer block. For example, to synthesize dimer block alkylphosphonates, condensation of a nucleoside 3'-alkylphosphoramidite with a 3'-protected nucleoside is carried out. For synthesis of dimer block phosphoramidates, alkylamine is added after H- phosphonate condensation of nucleotides. For synthesis of dimer block phosphorothioates, sulfurization follows condensation of nucleotides using an appropriate sulfur reagent after solution phase coupling of the protected monomeric nucleotides to yield a dimer. For preparing dimer block alkylphosphonothioates, an alkylphosphonoamidite is used in the same one pot reaction as described for dimer block phosphorothioates. This simple chemistry allows, for the first time, the synthesis of all possible dimer block methylphosphonothioates, of which only TT and TA were previously known, and promotes preparation of dimer blocks having 3'condensing groups. Thus, in a second aspect the invention provides novel dimer blocks comprising the nucleotides GG, GA, GT, GC, AG, AA, AT, AC, TG, TA TC, TT, CG, CA, CT or CC linked together by alkylphosphonate, phosphoramidate, phosphorothioate or alkylphosphonothioate linkages, and having various combinations of protective groups and condensing groups. These novel dimer blocks also give rise to a method of using such dimer blocks to assemble oligonucleotides containing alkylphosphonate, phosphoramidate, phosphorothioate, or alkylphosphonothioate linkages. Moreover, the dimer blocks according to the invention provide a method of using dimer blocks, for the first time, to assemble oligonucleotides having exclusively alkylphosphonate, phosphoramidate, phosphorothioate or alkylphosphonothioate internucleotide linkages, or mixtures thereof. Thus, in a third aspect, the invention provides methods of using dimer blocks to assemble oligonucleotides having alkylphosphonate, phosphorothioate, phosphoramidate or alkylphosphonothioate linkages or having combinations of these. It is an object of the invention to provide efficient methods that reduce total assembly time of oligonucleotides. It is a further object of the invention to provide efficient methods that reduce total solvent consumption required for oligonucleotide assembly. It is also an object of the invention to provide efficient methods that ease purification of oligonucleotides by increasing the yield of full length oligonucleotides. It is an additional object of the invention to provide efficient methods that reduce side reactions by reducing the exposure of partially assembled oligonucleotides to chemicals. Finally, it is an object of the invention to reduce overall cost of oligonucleotide assembly by allowing the use of inexpensive solution phase chemistry to achieve half of the total synthesis. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the synthesis steps for the phosphorothioate-containing dimer block 5 '-O-dimethoxytrityl- thymidine-3 '-O-methyl phosphorothioate-5'-0-N 4 -benzoy1-2 ' - deoxycytidine. In this scheme, (a) is anhydrous acetonitrile and tetrazole, (b) is Beaucage reagent, (c) is tetrahydrofuran and tetrabutyl ammonium fluoride, (d) is phosphorous trichloride and triazole in dichloromethane and 4- methylmorpholine, DMT is dimethoxytrityl, TBDMS is tert- butyldimethylsilyl and Et 3 NH + is triethylammonium. Figure 2 shows the synthesis steps for the phosphorothioate-containingdimerblock5'-O-dimethyoxytrityl- N 4 -benzoyl-2'-deoxycytidine-3'-O-methy.lphosphonothioate-5'-O- thymidine. In this scheme, (a) is acetonitrile and tetrazole, (b) is Beaucage reagent, (c) is pyridine acetic acid and hydrazine hydrate, (d) is phosphorous trichloride and triazole in dichloromethane and 4-methylmorpholine, DMT is dimethoxytrityl. Lev is levulinyl and Et 3 NH + is triethylammonium. Synthesis of the corresponding dimer block methylphosphonate is identical, except that I 2 is used in place of Beaucage reagent for step (b) . Figure 3 shows the synthesis steps for the phosphorothioate-containing dimer block S'-O-dimethoxytrityl- N 4 -benzoy1-2'-deoxycytidine-3'-O-cyanoethy1 phosphorothioate- 5'-O-thymidine. Identifiers are the same as in Figure 2. Figure 4 shows the synthesis steps for the phosphorothioate-containing dimer block 5'-O-dimethoxytrityl- N 6 -benzoyl-2 '-deoxyadenosine-3'-O-phosphorothioate 5 '-O-N 4 - benzoyl-2'-deoxycytidine. Identifiers are the same as in Figure 2. Figure 5 shows a dimer block according to the invention, wherein R, is a protective group such as dimethoxytrityl, mononmethoxytrityl or trityl, wherein L is an alkylphosphonate, phosphoramidate, phosphorothioate, or alkylphosphonothioate internucleotide linkage, R 2 is a hydroxyl group, or a β-cyanoethylphosphoramidite or H-phosphonate condensing group or a protective group such as levulinyl, t- butyl, or dimethylsilyl, Bl is G, A, T, or C, and B2 is G, A, T, or C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention relates to reagents and methods for assembling oligonucleotides. More particularly, the invention relates to the assembly of oligonucleotides having modified internucleotide linkages. In a first aspect, the invention provides new processes for making dimer blocks. Dimer blocks are dimeric nucleotides having modified internucleotide linkages and blocking groups at the 5'-hydroxyl. Preferred blocking groups include tert- butyldi-methylsilyl, dimethoxytrityl, levulinyl, mono ethoxytrityl and trityl groups. The 3' position of the dimer block may have a blocking group, a free hydroxyl, an H- phosphonate, a phosphotriester, or a β- cyanoethylphosphoramidite group. Preferred modified internucleotide linkages include phosphorothioate, alkylphosphonate and alkylphosphonothioate linkages. The modified linkage of the dimer block has an alkoxy or alkyl group. In a very general sense, the method of synthesizing dimer blocks according to the invention can be considered to be a method of synthesizing a dimer block having an alkylphosphonate, phosphoramidate, phosphorothioate or alkylphosphonothioate internucleotide linkage, the method comprising the steps of: (a) condensing together a first nucleoside derivative having a protective group at a 5' end and a condensing group at a 3' end with a second nucleoside derivative having a protective group at a 3' end and a hydroxyl group at a 5' end to form a dinucleotide derivative having a reduced internucleotide linkage, and (b) oxidizing the internucleotide linkage with an appropriate oxidizing agent to yield a dimer block alkylphosphonate, phosphoramidate, phosphorothioate or alkylphosphonothioate. The precise dimer block obtained, of course will depend upon the nature of the first nucleoside derivative and the oxidizing agent as shown in Table I, below. Table I SYNTHESIS OF DIMER BLOCKS Dimer Block Type First Nucleoside Derivative Oxidizing Agent alkylphosphonate nucleoside-3'-alkyl N,N di¬ iodine isopropyl phosphoramidite phosphoramidate nucleoside-3'-H phosphonate alkyl- or arylamine phosphorothioate nucleoside-3'-O-alkyl N,N, diisopropyl phosphoramidite sulfurizing or reagent nucleoside-3'-H-phosphonate alkyl- nucleoside-3'-alkyl N,N sulfurizing phosphonothioates diisopropyl phosphoramidite reagent In a first embodiment of this aspect of the invention, the method produces a dimer block having 5' and 3' blocking groups and a phosphorothioate internucleotide linkage. In this embodiment, the method comprises the steps of (a) joining together, by phosphoramidite or H-phosphonate chemistry, a nucleoside having a 5' blocking group and a nucleoside having a 3' blocking group, and (b) adding an appropriate sulfurizing agent, such as Beaucage reagent. Beaucage reagent (3H-1,2- benzodithiol-3-one 1,1-dioxide) is taught in U.S. Patent No. 5,003,097, the teachings of which are hereby incorporated by reference. Phosphorothioate dimer blocks can be made by using triester method also. In this case, 5'-DMT protected nucleoside will be first converted to an active intermediate by thiophosphorylating reagent and then coupled with 5'- hydroxyl nucleoside to give the phosphorothioate dimer. Such dimers can then be phosphitylated and used as synthons for assembly of oligonucleotide phosphorothioates and analogs. In a second embodiment of this aspect of the invention, the method produces a dimer block having a 5' blocking group, a 3' free hydroxyl group, and. a phosphorothioate internucleotide linkage. In this embodiment, the method comprises steps (a) and (b) of the first embodiment above, and further comprises the step of (c) deprotecting the 3'-hydroxyl group. This is achieved by the use of conditions selective for removal of the 3' protective group only. For example, if the 5' protective group is dimethoxytrityl, monomethoxytrityl or trityl, and the 3' group is tert-butyldimethylsilyl, then selective removal of the 3' group is obtained by treatment with tetrabutylammonium fluoride. Alternatively, if the 5' group is dimethoxytrityl, monomethoxytrityl or trityl and the 3' group is levulinyl, selective removal of the 3' group is obtained by treatment with hydrazine hydrate in pyridine/acetic acid. In a third embodiment of this aspect of the invention, the method produces a dimer block having a 5' blocking group, a 3' H-phosphonate group and a phosphorothioate internucleotide linkage. In this embodiment, the method comprises steps (a) , (b) and (c) of the first two embodiments described above and further comprises the step of (d) converting the free 3' hydroxyl group to an H-phosphonate group. Such conversion is described in detail in Example 5, below. In a fourth embodiment of this aspect of the invention, the method produces a dimer block having a 5' blocking group, a 3' β-cyanoethyl phosphoramidite group and a phosphorothioate internucleotide linkage. In this embodiment the method comprises steps (a) , (b) and (c) of the first two embodiments described above, and further comprises the step of (d) converting the free 3' hydroxyl group to a β-cyanoethyl phosphoramidite group. This conversion is described in detail in Example 8, below. Those skilled in the art will recognize that as an alternative to the third and fourth embodiments, dimer blocks having phosphotriester 3' groups can be prepared according to well known procedures. In additional embodiments of this aspect of the invention the method produces dimer blocks having a 5' blocking group, an alkylphosphonothioate, alkylphosphonate or phosphoramidate internucleotide linkage, and a 3' group that may be a blocking group, a free hydroxyl, an H-phosphonate group, a β-cyanoethyl phosphoramidate group, or a phosphotriester group. In these embodiments, the method is carried out exactly as described for the four embodiments above to produce dimer block alkyl- phosphonothioates, except that the starting material is a nucleoside alkylphosphoroamidite. Analogous dimer block alkylphosphonates are prepared in identical fashion to the dimer block alkylphosphonothioates, except that I 2 is used in place of the sulfurizing agent. Analogous dimer block phosphoramidates are prepared by H-phosphonate condensation followed by oxidation of the linkage with an alkyl- or arylamine. This first aspect of the invention offers a method of producing dimer block products that are useful as intermediates for assembling oligonucleotides having modified internucleotide linkages. The ability to produce these dimer blocks in a one pot reactions greatly simplifies their production. In a second aspect, the invention provides novel dimer block products having a 5' blocking group, a modified internucleotide linkage and a 3' group that may be a blocking group, a free hydroxyl, an H-phosphonate group, or in some cases a β-cyanoethyl phosphoramidite or phosphotriester group. The method for producing these dimer blocks is independent of the sequence of the nucleotides in the dimer block, thus allowing production of all possible dimer sequences containing alkylphosphonate, phosphoramidate, phosphorothioate or alkylphosphonothioate linkages, i.e.. GG, GA, GT, GC, AG, AA, AT, AC, TG, TA, TT, TC, CG, CA, CT, and CC. Such dimers are illustrated in Figure 5. In a third aspect, the invention provides a method of using dimer blocks to assemble oligonucleotides having modified internucleotide linkages. In this aspect, dimer blocks having modified internucleotide linkages (i.e.. phosphorothioate, alkylphosphonate, phosphoramidate, or alkylphosphonothioate) , are used to assemble oligonucleotides having such modified internucleotide linkages (dimer blocks having 5' blocking groups and 3' β-cyanoethyl phosphoramidite or H-phosphonate groups are used) . Synthesis is then carried out according to either the phosphoramidite or H-phosphonate approach. Support of oligonucleotide synthesis with dimer blocks can be soluble polymers as well as insoluble CPG and polymer beads. Those skilled in the art will recognize that this approach also allows the convenient synthesis of mixed phosphate backbone oligonucleotides, e.g. f oligonucleotides having both phosphorothioate and alkylphosphonothioate or phosphodiester and alkylphosphonothioate or phosphodiester and alkylphosphonothioate linkages or any combination of the modified linkages taught herein with each other or with phosphodiester linkages. The method according to this aspect of the invention provides several advantages over monomeric synthesis of oligonucleotides. First, since half as many assembly cycles are required, the total assembly time is reduced by half, which for large scale synthesis can be a saving of 12 hours or more for a single oligonucleotide. This reduction in time also results in fewer side reactions, since partially assembled oligonucleotides are exposed to chemicals for a shorter time. The method also facilitates purification of oligonucleotides by increasing the proportion of full length oligonucleotides, since that proportion varies inversely with the number of cycles performed. Finally, the method reduces cost of synthesis by cutting solvent consumption by half and by allowing one half of the total synthesis to be carried out using inexpensive solution phase chemistry. The present method extends these advantages to oligonucleotides having exclusively phosphorothioate, alkylphosphonate, phosphoramidate, or alkylphosphonothioate linkages as well as to oligonucleotides having any combination thereof. The following examples are intended to further illustrate certain preferred embodiments of the methods according to the invention, and are not intended to be limiting in nature. Example 1 Solution Phase synthesis Of 5 , -o-dimethoxytrityl-thymidine- 3'-o-methyl phosphorothioate-5'-0-N-benzoyl-2'-deoxycytidine The synthesis steps for this protected dimer block for synthesis of phosphorothioate containing oligonucleotides are shown in Figure 1. A mixture of 5'-0-dimethoxytrityl-thymidine-3'-O-methyl N,N-diisopropyl phosphoramidite, 1 , (1.4 g, 2 mmol) and N 4 - benzoyl-3'-0(tert-butyldimethylsilyl)-2'-deoxycytidine, 2., (0.88g, 2 mmol) was dissolved in anhydrous acetonitrile (25 ml) and a solution of tetrazole (0.45 M, 10 ml) was added. The reaction mixture was stirred at room temperature for 15 min. Beaucage reagent (0.6 g in anhydrous acetonitrile 15 ml) was added and the mixture was further stirred for 15 min. The reaction mixture was evaporated to remove most of the acetonitrile under reduced pressure. The crude reaction product was extracted with dichloromethane and washed with brine. The organic layer was dried over Na 2 S0 4 and evaporated to dryness to obtain 3.. Product 2 was re-dissolved in tetrahydrofuran (16 ml) and treated with a 1 M solution of tetrabutylammonium fluoride (3 ml, THF) for 15 min. The reaction mixture was evaporated to almost dryness and partitioned between dichloromethane and water. The organic layer was dried over Na 2 S0 4 and evaporated to a small volume. The product was purified by column chromatography using silica gel (2.5 x 20 cm). The dimer block product, 4_, was eluted with 0-7% methanol in dichloromethane (0.5% pyridine); obtain 1.3 g (70% yield); not optimized. 31 P NMR = 70.06. Example 2 Solution Phase Synthesis Of S'-O-dimethoxytrityl-l^-benzoyl- 2 '-deoxycytidine-3'-O-methyl phosphorothioate-5'-o-thymidine The synthesis steps for this dimer block for synthesis of phosphorothioate-containing oligonucleotides are shown in Figure 2. A mixture of N 4 -benzoyl 5'-0-dimethoxytrityl-2'- deoxycytidine-3'-O-methyl N,N-diisopropyl phosphoramidite 6 (1.6 g, 2 mmol) and 3'-0-levulinyl-thymidine, 1_, (0.68 g, 2 mmol) was dissolved in anhydrous acetonitrile (25 ml) and a solution of tetrazole (0.45 M, 10 ml) was added. The reaction mixture was stirred for 15 minutes at room temperature. Beaucage reagent (0.6 g in anhydrous acetonitrile 15 ml) was added and the reaction mixture was further stirred for 15 minute The reaction mixture was then evaporated to remove most of the solvent. The crude reaction product was extracted with dichloromethane and washed with brine to obtain 8_. Dichloromethane was evaporated, the solid residue was re- dissolved in 20 ml pyridine and mixed with 20 ml of 1 M hydrazine hydrate solution in pyridine/acetic acid (3/2) . The reaction mixture was stirred for 5 min. The reaction mixture was then cooled on an ice-bath and 4 ml acetyl acetone was added to quench the excess amount of hydrazine hydrate. The mixture was evaporated to a small volume and then directly applied to silica gel column chromatography (2.5 x 25 cm). The dimer block product, 9_, was eluted by using 0-7% methanol in dichloromethane (0.5% pyridine) to obtain 1.25 g (67% yield). 31 P NMR = 69.86. Example 3 SolutionPhaseSynthesisOf 5'-0-dimethoxytrityl-N 4 -benzoyl-2'- deoxycytidine-3'-o-cyanoethylphosphorothioate-5'-O-thymidine The synthetic steps for this dimer block for synthesis of phosphorothioate-containing oligonucleotides are shown in Figure 3. A mixture of N 4 -benzoyl-5'-0-dimethoxytrityl-2'- deoxycytidine-3 '-O-β-cyanoethyl N,N-diisopropyl phosphoramidite, Iλ, (1-7 g, 2 mmol) and 3'-0-levulinyl thymidine, Z, (0.68 g, 2 mmol) was dissolved in anhydrous acetonitrile (25 ml) and a solution of tetrazole (0.45 m, 10 ml) was added. The reaction mixture was stirred for 15 minutes at room temperature. Beaucage reagent (0.6 g in anhydrous acetonitrile 15 ml) was added and the reaction was continued for a further 15 minutes. The reaction mixture was evaporated to remove most of the solvent. The product, 12. was purified and deprotected with hydrazine hydrate by the same procedure as described for product , in Example 2. Product 12 / the dimer block was obtained after purification of silica gel column to obtain 1.2 g (59% yield) . 31 P NMR = 67.80. Example 4 SolutionPhaseSynthesis Of 5'-0-dimethoxytrityl-N 6 -benzoyl-2'- deoxyadenosine-3'-o-phosphorothioate 5'-0-N 4 -benzoyl -2'deoxycytidine The synthetic steps for this dimer block for synthesis of phosphorothioate-containing oligonucleotides are shown in Figure 4. A mixture of N 4 -benzoyl-5'-0-dimethoxytrityl-2'- deoxycytidine-3'-O-methyl N,N-diisopropyl phoshoramidite, 15. (4 g, 5 mmol) and 3'-0-levulinyl-N 6 -benzoyl-2'-deoxyadenosine, 16. (2 g, 4.4 mmole) was dissolved in anhydrous acetonitrile (18 ml) and a solution of tetrazole (0.45 m, 22 ml) was added. The reaction mixture was stirred for 30 minutes at room temperature, then treated with Beaucage reagent (1.4 g in anhydrous acetonitrile 25 ml) for 15 minutes. The reaction mixture was then evaporated to remove most of the solvent. The crude reaction product was extracted with dichloromethane and washed with brine. Dichloromethane was evaporated to obtain a solid product, 12 / which was then redissolved in 40 ml pyridine, and 40 ml of 1 M hydrazine hydrate solution in pyridine/acetic acid (3/2) was added. After 7 minutes, the reaction was quenched with ice and the product was extracted with dichloromethane, then washed with water. The organic layer was dried over sodium sulfate and then co-evaporated with toluene to dryness. The mixture was re-dissolved in a small volume of dichloromethane and applied to silica gel column chromatography (5 x 12 cm) . The dimer block product, 18. was eluted by using 0-7% methanol in dichoromethane (0.5% pyridine) to obtain 3.8 g (79% yield). 31 P NMR = 69.84, 69.89. Example 5 H-phosphonate Derivatization of Dimer Blocks 0.62 ml phosphorous trichloride (7 mmol) was added to 4.9 g triazole (70 mmol) in dichloromethane (60 ml) , followed by 4-methylmorpholine (7.8 ml, 70 mmol) . The mixture was stirred at room temperature for 30 minutes and then cooled with an ice bath. A solution of fully protected dimer block, 4., 5'- dimethoxytrityl-thymidine-3'-O-methyl phosphorothioate-5'-O-N 4 - benzoyl-2'-deoxycytidine, prepared according to Example 1, (1.3 g 1.4 mmol) in dichloromethane (20 ml) was added dropwise and then the mixture was stirred at room temperature for 30 min. The reaction was quenched with 1 M TEAB buffer (pH 7.5) . The mixture was extracted with dichloromethane and washed with 1 M TEAB once. The organic extract was evaporated to obtain a solid residue and further purified on silica gel column chromatography (2.5 x 20 cm), eluted by 0-10% methanol in dichloromethane (0.2% TEA) to obtain 0.97 g (63% yield of H- phosphonate derivatized dimer block 5_. 31 P NMR = P v 70.0, P m 3.8. H-phosphonate derivatives of dimer blocks 9., 12, and 18. prepared according to Examples 2, 3 and 4, respectively, were synthesized by the above procedures to obtain H-phosphonate derivatized dimer blocks 10, 2Λ and 19 respectively. Example 6 Use of H-phosphonate Derivatized Dimer Blocks To Assemble Phosphorothioate-containing Oligonucleotides Assembly of oligonucleotides was carried out on 1 μmole scale using an H-phosphonate approach as taught in U.S. Patent No. 5,149,798 the teachings of which are hereby incorporated by reference, on an automated synthesizer (Millipore Model 8700) . Dimer block H-phosphonate Q. was dissolved in acetonitrile/pyridine (1:1; v/v) mixture 10 mM solution and stored in the U reservoir of the DNA synthesizer. The following two oligonucleotides were synthesized: Oligonucleotide 1 5' T C C T T C T Oligonucleotide 2 5' T C C T T C T Oligonucleotide 1 was synthesized using nucleoside H- phosphonates as synthons. For assembly of oligonucleotide 2, the first two couplings were carried out using nucleoside H-phosphonates, followed by a coupling with dimer block H- phosphonate 10., prepared according to Examples 2 and 5. The last two couplings were carried out using nucleosides H- phosphonates. At the end of assembly of both oligonucleotides 1 and 2, the CPG bound oligonucleoside H-phosphonate intermediate was oxidized with 5% elemental sulfur in triethylamine/ pyridine/carbon disulfide (1:10:10; v/v/v) to convert H- phosphonate linkages to phosphorothioate linkages. Deprotection was carried out in concentrated ammonia at 55° C for 10 hours. The products were analyzed by high performance capillary electrophoresis, ion exchange HPLC and polyacrylamide gel electrophoresis. Both oligonucleotide l and 2 gave similar results. After the successful assemblies of oligonucleotide 2, using one dinucleotide H-phosphonate 5 unit, the followng oligonucleotide 3, a 25 mer, was synthesized using nucleoside H-phosphonates and dimer block H-phosphonate, 4 ./ as prepared in Examples 1 and 5. Oligonucleotide 3: 5' C T C T C G C A C C C A T C T C T C T C C T T C T 3' At the underlined positions, coupling was carried out using dimer block H-phosphonate 4 . . The rest of the sequence was assembled using nucleoside H-phosphonates. After the assembly of the above sequence, CPG bound oligomer was oxidized using 5% elemental sulfur in triethylamine/pyridine/ carbon disulfide (1:10:10; v/v/v) to convert H-phosphonate linkages to phosphorothioate linkages. Methoxyl groups were removed by treatment with thiophenol. Deprotection of oligonucleotide was then carried out in concentrated ammonium hydroxide at 55° C for 10 hours. The oligonucleotide was analyzed by high performance capillary electrophoresis, ion exchange HPLC and polyacrylamide gel electrophoresis. Example 7 Solution Phase Synthesis Of S'-O-dimethoxytrityl- thymidyl-3'-O-methyl phosphonothioate-5'-O-thymidine The synthetic steps for this dimer block for phosphoramidite synthesis of alkylphosphonothioate-containing oligonucleotides are shown in Figure 5. A mixture of 5'-O-dimethoxytrityl-thymidine-3'-methyl N,N-diisopropyl phosphoramidite (0.5 g, 0.74 mmol) and 3'-0- levulinyl-thymidine (0.25 g, 0.7 mmol) was dissolved in anhydrous acetonitrile (7 ml) and a solution of tetrazole (0.45 M, 3.5 ml) was added. The reaction mixture was stirred for 30 minutes at room temperature, treated with Beaucage reagent (0.29 g in anhydrous acetonitrile, 10 ml) for 15 minutes and then evaporated to remove most of the solvent. The crude reaction product was extracted with dichloromethane and washed with brine. After removal of dichloromethane, the material was redissovled in 7.5 ml pyridine, and 7.5 ml 1 M hydrazine hydrate solution in pyridine/acetic acid (3/2) was added. After 5 minutes, the reaction mixture was cooled in an ice-bath and 2 ml acetyl acetone were added to quench the excess amount of hydrazine hydrate. The mixture was evaporated to a small volume and then directly loaded to a silica gel column (2.5 x 25 cm) . The dimer block product, 22 f was eluted by using 0 to 5% methanol in dichloromethane (0.5% pyridine) to obtain 0.5g (82% yield). 31 P NMR = 97.7, 99.0. Example 8 β-cyanoethyl Phosphoramidite Derivatization Of An Alkylphosphonothioate-Containing Dimer Block To a solution of fully protected dimer block 2_2, 5'-0- dimethoxytrityl-thymidyl-3'-O-methyl phosphorothionate-5'-o- thymidine prepared according to Example 7, (0.5 g, 0.58 mmol) in anhydrous THF (5 ml) , was added N,N-diisopropyl ethylamine (0.42 ml, 2.4 mmol) and then β-cyanoethyl N,N-diisopropyl- chlorophosphoramidite (0.27 ml, 1.2 mmol) . The reaction mixture was stirred for 30 minutes under nitrogen, at room temperature and silica gel TLC (ethyl acetate with 10% triethylamine) showed that the reaction was completed. After filtration of the precipitates, the reaction mixture was evaporated. The product was extracted with ethyl acetate and washed with sodium bicarbonate and brine. After drying with anhydrous sodium sulfate, the β-cyanoethyl phosphoramidite derivatized dimer block product, 22, was evaporated to a small volume and then precipitated in hexane (-78° C) . Obtain 0.51 g (81% yield). 31 P NMR = P v 97.7, 99.0; P πι 149.3, 149.7. This same procedure was used to prepare β-cyanoethyl phosporamidite derivatives of phosphorothioate-containing dimer blocks. The phosphorothioate-containing dimer blocks 9_, 12 and 4., as prepared in Exampled 2, 4 and 1, respectively, were used in the procedure above to obtain the β-cyanoethyl phosphoramidite derivatized dimer blocks 2 , 2_5 and 26. respectively. Example 9 Use Of An Alkylphosphonothioate-containing Dimer Block Phosphoramidite To Assemble Alkylphosphonothioate Containing Oligonucleotides Assembly of oligonucleotides was carried out in 1 μmol scale using a standard phosphoramidite approach (see, e.g. , U.S. Patent No. 5,047,524, the teachings of which are hereby incorporated by reference) and iodine oxidation on an automated synthesizer (Millipore Model 8700) . Methylphosphonothioate dimer phosphoramidite 23 was dissolved in anhydrous acetonitrile (25 mg/ml) and stored in the U reservoir of the DNA synthesizer. The following oligonucleotides were synthesized: Oligonucleotide 4 5' T T T T T T T T Oligonucleotide 5 5' T T T T T T T T For assembly of oligonucleotide 4, the first two couplings were carried out using protected thymidine phosphoramidite (with iodine oxidation) , followed by coupling with methylphosphonothioate dimer (without oxidation) . The last three couplings were carried out using protected thymidine phosphoramidiate (with iodine oxidation) . At the end, deprotection was done with concentrated ammonia at room temperature for one hour. The product was analysed by high performance capillary electrophoresis and reverse phase HPLC. On reversed phase HPLC, retention time of oligonucleotide 4 was 18.96 minutes, compared to oligonucleotide 5, which had a retention time of 17.11 minutes. The increase in retention time of oligonucleotide 5 is an indication of increased hydrophobicity of oligonucleotide 5 due to its one methylphosphonothioate linkage. Example 10 Solution Phase Synthesis Of 5'-Dimethoxytrityl-thymidyl- 3 i -o-methyl phosphonate-5'-O-thymidine A mixture of 5'-O-dimethoxytrityl-thymidine-3'-methyl N,N-diisopropyl phosphoramidite (0.5g, 0.74 mmol) and 3'-0- levulinyl-thymidine (0.17g, 0.5 mmol) was dissolved in anhydrous acetonitrile (7 ml) and a solution of tetrazole (0.45 M, 3.5 ml) was added. The reaction mixture was stirred for 30 minutes at room temperature and then partitioned between dichloromethane and water. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. To the residue, 10 ml of 2.5% iodine solution in 2.6- lutidine/water/THF (1:1:2) was added and stirred for 15 minutes. The mixture was poured into sodium bisulfite solution and extracted with dichloromethane. After removal of solvent, the product was re-dissolved in 5 ml pyridine and 5 ml 1 M hydrazine hydrate solution in pyridine/acetic acid (3:2) was added. After 7 minutes, the reaction mixture was quenched with ice and extracted with dichloromethane and water. The mixture was dried over anhydrous sodium sulfate and evaporated to dryness. The dimer block product was purified by silica gel column (2.5 x 25 cm) using 0-5% methanol in dichloromethane (0.5% pyridine) to obtain 0.25 g (60%) 31 PNMR = 32.4.33. All other sequences of dimer block alkylphosphonates can be prepared in the same manner. To convert these dimer blocks having 3'hydroxyl groups to their H-phosphonate or β- cyanoethylphosphoramidite derivatives, the 3' hydroxyl dimer blocks are processed according to Examples 5 or 8 respectively. Example 11 Solution Phase Synthesis Of 5'-0-dimethoxytrityl-thymidyl -3'-O-n-butyl phosphoramidate-5'-0-thymidine A mixture of 5'-0-dimethoxytrityl-thymidine-3'-H- phosphonate triethylammonium salt (0.5g, 0.7 mmol) and 3'-0- levulinyl-thymidine (0.2g, 0.6 mmol) was dissoved in 6 ml anhydrous pyridine/acetonitrile (1:1) and 1-adamantane carbonyl chloride (0.7 g, 0.5 mmol) in the same solvent (6 ml) was added. The reaction mixture was stirred for 15 minutes at room temeprature and then partitioned between dichloromethane and water. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. To the residue, 10 ml of 2% n-butylamine solution in carbon tetra-chloride was added and stirred for 20 minutes. The product was extracted with dichloromethane and washed with water. After removal of solvent, the residue was redissovled in 5 ml pyridine and 5 ml 1 M hydrazine hydrate solution in pyridine/acetic acid (3:2) was added. After 7 minutes the reaction mixture was quencehd with ice and extracted with dichlormethane and water. The mixture was dried over anhydrous sodium sulfate and evaporated to dryness. The n-butyl phosphoramidate dimer block product was purified by silica gel column (2.5 x 25 cm) using 0-5% methanol in dichloromethane (0.5% pyridine) to obtain 0.4 g (74%) 31 P NMR = 10.9. All other sequences of dimer block phosphoramidates can be prepared in the same manner. The nature of the amide can be changed by substituting another alkylamine or arylamine for n-butylamine. To convert these dimer blocks having 3' hydroxyl groups to their H-phosphonate or phosphoramidite derivatives, the 3' hydroxyl dimer blocks are processed according to Example 5 or 8 respectively.

Claims: We claim: 1. A method of synthesizing a dimer block having an alkylphosphonate, phosphoramidate, phosphorothioate or alkylphosphonothioate internucleotide linkage, the method comprising the steps of: (a) condensing together a first nucleoside derivative having a protective group at a 5' end and a condensing group at a 3' end with a second nucleoside derivative having a protective group at a 3' end and a hydroxyl group at a 5' end to form a dinucleotide derivative having a reduced internucleotide linkage, and (b) oxidizing the internucleotide linkage with an appropriate oxidizing agent to yield a dimer block alkylphosphonate, phosphoramidate, phosphorothioate or alkylphosphonothioate. 2. The method according to claim 1, wherein the internucleotide linkage is an phosphoramidate linkage, the first nucleoside derivative is a 5' protected nucleoside having an H-phosphonate condensing group, and the oxidizing agent is an alkylamine or arylamine. 3. The method according to claim 1, wherein the intemucleoside linkage is a alkylphosphonate linkage, the first nucleoside derivative is a 5' protected nucleoside-3' alkyl N,N diisopropyl phosphoramidate and the oxidizing agent is iodine. 4. The method according to claim 1, wherein the internucleotide linkage is an alkylphosphonothioate linkage, the first nucleoside derivative is a 5' protected nucleoside- 3'-alkyl N,N diisopropyl phosphoramidate and the oxidizing agent is a sulfurization reagent. 5. The method according to claim 1, wherein the internucleotide linkage is a phosphorothioate linkage, the first nucleoside derivative is a 5' protected nucleoside-3'-0- alkyl N,N diisopropyl phosphoramidate or a nucleoside 3'-H- phosphonate and the oxidizing agent is a sulfurization reagent. 6. A dimer block as shown in Figure 5 wherein P is a protective group, L is an alkylphosphonate, phosphoramidate, phosphorothioate, or alkylphosphonothioate group, G is a protective group, a hydroxyl, or a phosphoramidite or H- phosphonate group, Bl is G,A, T or C and B2 is G,A, T, or C. 7. The dimer block according to claim 6, wherein L is an alkylphosphonate group. 8. The dimer block according to claim 6, wherein L is a phosphoramidate group. 9. The dimer block according to claim 6, wherein L is a phosphorothioate group. 10. The dimer block according to claim 6, wherein L is an alkylphosphonothioate group. 11. The dimer block according to claim 6, wherein G is a phosphoramidite group or an H-phosphonate group. 12. A method of using dimer blocks to assemble oligonucleotidescontainingphosphoramidate, alkylphosphonate, phosphorothioate and/or alkylphosphonothioate internucleotide linkages, the method comprising the steps of: condensing a dimer block according to claim 11 with a free 5' hydroxyl group on a growing oligonucleotide chain or with a free hydroxyl group attached to a solid support.

Inventors: AGRAWAL SUDHIR, IADAROLA PATRICIA L, TANG JIN-YAN, AGRAWAL, SUDHIR, IADAROLA, PATRICIA, L., TANG, JIN-YAN
Applicants: AGRAWAL SUDHIR, HYBRIDON INC, IADAROLA PATRICIA L, TANG JIN YAN, AGRAWAL, SUDHIR, HYBRIDON, INC., IADAROLA, PATRICIA, L., TANG, JIN-YAN
Application year: 1994
Main classification: C07H21
IPC codes: C07H21